Nonclassical Crystallization and Core–Shell Structure Formation of Ibuprofen from Binary Solvent Solutions

Liquid–liquidphase separation (LLPS) or dense liquid intermediates during the crystallization of pharmaceutical molecules is common; however, their role in alternative nucleation mechanisms is less understood. Herein, we report the formation of a dense liquid intermediate followed by a core–shell structure of ibuprofen crystals via nonclassical crystallization. The Raman and SAXS results of the dense phase uncover the molecular structural ordering and its role in nucleation. In addition to the dimer formation of ibuprofen, which is commonly observed in the solution phase, methyl group vibrations in the Raman spectra show intermolecular interactions similar to those in the solid phase. The SAXS data validate the cluster size differences in the supersaturated solution and dense phase. The focused-ion beam cut image shows the attachment of nanoparticles, and we proposed a possible mechanism for the transformation from the dense phase into a core–shell structure. The unstable phase or polycrystalline core and its subsequent dissolution from inside to outside or recrystallization by reversed crystal growth produces the core–shell structure. The LLPS intermediate followed by the core–shell structure and its dissolution enhancement unfold a new perspective of ibuprofen crystallization.

. Experimental flow of preparation of ibuprofen precipitate and core-shell structure. The X-ray powder diffraction patterns were recorded on a PANalytical X'Pert PRO MPD Alpha 1 using Cu Kα radiation (λ = 1.5406 Å) at 45kV over the 2θ range of 5-60° with step size 0.02° and 20 s per step. The KrystalShaper (Version 1.5.0) was used to create the crystals morphology.

S1.2. Raman spectra of dense liquid and solid samples
Renishaw inVia™ confocal Raman microscope was used to record the vibrational spectra for the IBU dense phase and IBU precipitate. The dense phase spectra were compared with the solid to investigate the structural order in the former case. An emission wavelength of 785 nm (nIR) was selected as an excitation source in a continuous-wave gas laser. The dense phase in LLPS was carefully collected and placed under the microscope and the objective lens (x63) of Leica imaging microscope probe was immersed to collect the spectra. A noncontact objective lens 20x was focused and spotted the crystalline samples to record the solid spectra. The spectra were collected in the region 100-3200 cm -1 with 10 s exposure time and three accumulations. The 830 lines/mm grating was used to collect the whole spectra region and 1800 lines/mm grating was used to collect the mid and low-frequency region. The Wire 5.3 software was used to collect data.

S1.3. Scanning electron microscope
The morphology of the samples was examined using Zeiss Sigma VP scanning electron microscope (SEM) with an accelerating voltage 5 kV and InLens detector. The samples were fixed on a metal stud using two-sided carbon tape and the samples were sputtered approximately 10 nm using Au Pd using Leica EM AC600 sputtering device to avoid the conductivity issues. Image J software was used to analyze the particle size from SEM images.

S1.4. SEM interfaced focused ion beam (FIB)
The cross-sectional image of the core-shell structure was obtained using SEM interfaced focused ion beam experiment. The JEOL JIB-4700F was used with an accelerating voltage of 30 kV and Ga+ ions were employed to cut the core-shell structure. The particles were mounted on an aluminum stud and Au Pd was coated similar to SEM measurement. To get a smooth cut, the sample under vacuum was coated with a 1 µm layer of platinum. Different beams were used for fine and coarse milling of the samples. Since the angle between ion beam and to the SEM detector is 45°, the cut sample was tilted so, and their cross-sections were imaged.

S1.5. TGA/STA Thermal analysis
The simultaneous thermal analyzer NETZSCH STA 449 F3 Jupiter & QMS Aeolos Quadro was used to measure the mass change and thermal effect of the core-shell structure and precipitate. The temperature range was in the region 40-600 ℃ at a heating rate of 10 K/min in the crucible of Al 2 O 3 85 µl under a flow of helium purge gas at the rate of 70 mL/min.

S1.6. Small angle X-ray scattering of liquid and solid samples
The different IBU liquid samples such as supersaturated solution after cooling and IBU dense phase and other solid samples (IBU single crystals and IBU precipitate) were investigated using SAXS technique. A Xenoxs Xeuss 3.0 interfaced with Full travel in-vacuum motorized HPC detector was used. The sample to detector distance was 0.5 m and the X-ray wavelength was λ = 1.542 Å. For the liquid samples, the supersaturated solution immediately after cooling and dense phase collected from LLPS were separately filled in microtubes using micro syringe and both ends were sealed completely. The SAXS was performed for all the samples at a time. A SasView 5.0.4 software was used to fit the data. The SAXS data fit were performed by Guinier Porod model using following mathematical equation 1 . The model gives a good estimation of the particle size (radius of gyration, Rg) and diverse shape of the scattering particles (Porod exponent). For instance, the Porod exponent equal to one implies the rod-like shape of the particles.
where I(Q) is the intensity, G Guinier factor, Q-difference between the incident and the scattered beam and Rg radius of gyration, Q1 transition point and d Porod exponent For the dense phase, as the data does not fit well for the Guinier-Porod model, we choose Guinier model below to fit well in the low q region and this model is often used for monodispersed solution.

S1.7. Dissolution test
Dissolution test for the as purchased, single crystals, core-shell structure and precipitate of IBU was performed by following The United States Pharmacopoeia rotating disk method in phosphate buffer (pH 7.2, 900 mL) at 37 °C, using Distek dissolution system 2100B apparatus. The rotating speed was at 100 rpm and 100 mg of pelletized IBU was used for the dissolution. The drug sample was compressed at 1 ton for 90 s. At the fixed time intervals of 1, 2, 5, 10, 15, 20, 30, 45, 60, 120, 240, 360 min, the release medium of 5 mL sample was withdrawn, and an equal amount of fresh dissolution medium was added. Withdrawn samples were filtered through 0.45 μm membrane filter, and the concentration was quantified spectrophotometrically at the excitation of 221 nm wavelength using a UV-VIS spectrophotometer (Lambda 40, Perkin Elmer). The percentage of drug release versus time was plotted to realize the impact of the dissolution profile of different samples.

S2.1. Powder X-ray diffraction
The X-ray powder diffraction patterns of as purchased racemic (±) IBU and IBU precipitate are presented in Fig. S3. The diffraction peak observed at the angle 2θ=6.12 (d=14.41) is the characteristic peak that differentiate the racemic (±) IBU from S(+)-IBU enantiomer. For the as purchased IBU, the growth along (110) direction is high, while for the IBU precipitate other planes such as (210), (211), and (112) are higher. There are no significant differences in peak positions or line shapes other than the intensity difference in the diffraction pattern. . Powder X-ray diffraction pattern of as purchased and precipitate of racemic (±) ibuprofen. As the peak intensity of (100) is significantly high and suppressing the height of other peaks, the intensity scale has been restricted to 40000 (y-axis) in the as purchased sample.

S2.2. Raman spectra of dense and solid phases
The Raman spectra of IBU in the range of 100-3200 cm -1 is presented in Fig. S4 and functional groups assigned for the vibrations are listed in Table S1. The molecular structure of IBU with atom numbering is presented in Fig. S5. The C-H vibrations are observed in the higher frequency region of 2800-3100 cm -1 and the vibrations associated with benzene (3059 cm -1 ) have not shown any significant changes however major redshift (16 cm -1 and 3 cm -1 ) respectively has been observed for the C-H associated to chiral center (3036 cm -1 ) and CH 2 in methylpropyl (2869 cm -1 ) for dense phase compared with the solid 2 . The redshift associated with CH 2 does not occur in hydrogen bond alone (dimer) compared with solvation shell elsewhere 3 . These red shifts due to methyl group that are not involved in hydrogen bonding support our claim that the vibrational changes are not only due to dimer formation in the IBU.

S2.3. SEM images of HPMC and Pluronic, PVP and PVA influences on IBU.
The HPMC is a neutral polymer with abundant hydrogen bond donors and acceptors in the bulk structure (growth inhibitor). Similarly, Pluronic-F127 is copolymer which can be used as crystal size inhibitor. The solvent ratio used for the precipitate is 4 and 1.5 for the slow evaporation. The effects induced by HPMC and Pluronic-F127 are trivial towards the coreshell formation. The HPMC produces rough plate like and chunky particles with irregular shape. Since our aim is to control the particle formation at the initial stage of crystallization, but not to control the morphology, we used limited amount of additives for e.g. 0.3 wt % of HPMC (Fig.S6 a). The pH=4.83 environment has not impacted the morphology (Fig.S6 b) as it looks similar to Fig.S6a. The dense phase was harvested and dried before SEM measurement. The crystals are highly porous, and the long polymer chain has produced IBU in the form of fiber attached together. Similar to IBU-HPMC-Slow evaporation, the addition of Pluronic-F127 has not produced any core-shell structure. The pH (pH=5.35) environment along with Pluronic-F127 (Fig.S6e) produces aggregation which is different from addition of Pluronic-F127 alone as presented in Fig.S6d. The IBU-Pluronic-Slow evaporation (Fig.S6f) produces crystals similar as IBU-HPMC-Slow evaporation (Fig. S6c).  The PVP has a hydrogen bonding acceptor group without a donor. The PVA is amphiphilic and non-ionic polymer with hydrogen bonding donating and accepting groups whereas in PAA, there is an acid group similar to IBU with the presence of a donor group double that of the acceptor. The precipitate of IBU without the addition of additives and with PVP and PVA is presented in Fig. S8 (a-i). The precipitate shows chunky and rod-like morphology with a rough surface. The final products from the slow evaporation are porous and the magnified view shows the texture like structure ( Fig.S8 (b-c)). The use of 1.5 wt % of PVP without pH and with pH=6.3 does not show any difference (Fig.S8 (d-e)) compared to precipitate of IBU without and with HPMC and Pluronic-F127 (Fig. S6). However, slow evaporation of 1.5 wt % PVP added IBU solution has grown crystals with clear facets in 3 days (Fig. S8f). The formation of coreshell morphology is absent in the case of precipitation and it was further confirmed by raising the PVA amount to 0.3 wt % without pH (Fig. S8g) and with pH= 5.08 (Fig. S8h). The slow evaporation of 1.5 wt % PVA added IBU solution with pH=6.25 has grown chunky crystals (Fig.  S8i) without clear facets as shown in Fig.S8f.      The Porod exponent is 3<P<4 for the crystals which implies the rough surface. For other solid samples, the value is greater than four, which means the particles are smooth or the particle have somewhat diffused boundary. In case of diffused boundary, the electron density profile in the scattering center is not same and thus shows density contrast between the two particles. IBU single crystals IBU precipitate IBU 0.3 wt% PAA core-shell structure IBU 0.75 wt% PAA core-shell structure Fig. S14. The comparison plot of SAXS for the IBU PAA core-shell structure 0.75 wt % (b), IBU PAA core-shell structure 1.5 wt % (c) and IBU PAA precipitate (d).